The invention is in the field of diagnostic imaging and therapeutics. It relates to novel metal chelates containing metal species bound by 2-pyrrolylthione core in N2S2 fashion. Methods for the preparation of the chelate complexes are provided. The invention also provides pharmaceutical compositions comprising the metal chelate, and the use of this composition as a diagnostic imaging or therapeutic agent. Kits comprising the compounds and compositions of the invention are also provided.
The art of diagnostic imaging employs contrasting agents that in binding or accumulating site-specifically within the body help to resolve the image of diagnostic interest.
For example, renography using radiotracers is the method of choice that allows the determination of both total and differential renal function and the detection of obstructions in urine flow. For this purpose, a composition comprising the radiopharmaceutical, such as an injectable liquid, is administered to the patient. By means of a suitable detection apparatus, such as a camera for detecting radiodecay, images can be obtained by recording the emitting radiation. Thus the organ or the pathological process in which the radiopharmaceutical has been incorporated may be visualized.
Among the oldest and most widely employed techniques for renal function evaluation are the renal clearance methods; the most fundamental of which is directed toward determination of glomerular filtration rate (GFR). In addition, the clearance of compounds that undergo extensive tubular excretion in addition to filtration allow the evaluation of functional tubular mass and the estimation of effective renal plasma flow (ERPF).
The standard for ERPF determination is p-aminohippurate (PAH), of which approxymately 90% is extracted from the renal arterial plasma in a single pass through the renal parenchyma. An I-131 labeled structural analog, ortho-iodohippurate ([131I]OIH; Hippuran), has been the clinical standard for the past 30 years. Although OIH yields a good approximation of renal plasma flow, the 364 keV photon energies of I-131 results in poor spatial resolution and the emission of beta partcles increases the radiation dose to the patient. Labeling OIH with I-123 results in a better imaging agent, but the availability and prohibitively high cost of I-123 limits the use of this compound.
Because of the favorable physical properties, widespread availability, and low cost of technetium-99m (Tc-99m), this radionuclide continues to be the most attractive candidate for the formulation of diagnostic radiopharmaceuticals to be used in scintigraphic gamma-imaging studies in patients (Jurisson et al., xe2x80x9cCoordination compounds in nuclear medicinexe2x80x9d. Chem.Rev. 93:1137-1156 (1993)).
[99mTc]TcO(glucoheptonate)2, Glucoscan, also known as Technescan, is an early kidney imaging agent, the precise structure of which has never been determined. While this complex is no longer widely used as an imaging agent; however, it is regularly used as a precursor for the synthesis of other Tc(V) species via ligand exchange.
[99mTc]Tc-diethylenetriaminepentaacetic acid (or [99mTc]Tc-DTPA) has received regulatory approval for use as a kidney imaging agent. The structure of this complex has not yet been determined unequivocally, and it is unclear as to whether the complex contains technetium in the IV or V oxidation state. This radiopharmaceutical has very limited clinical applications.
Early attempts to create a Tc-99m-based renal imaging agent focussed on the diamine dithiolate (DADT) ligands. 99mTc-N,Nxe2x80x2-bis(mercaptoacetyl)-2,3-diaminopropanoate (99mTc-CO2DADS) (Fritzberg et al., xe2x80x9cSynthesis and biological evaluation of Tc-99m-N,Nxe2x80x2-bis(mercaptoacetyl)-2,3-diaminopropanoate: A potential replacement for [I-131]-o-iodohippuratexe2x80x9d 23:592-598 (1982)) has a favorable renal clearance profile, but this compound consists of stereoisomers with different rates and specificities for renal clearance. Therefore, HPLC separation of the desired stereoisomer is required, which makes routine preparation inconvenient.
The para-aminohippuric (PAH) acids have been found to be almost completely extracted from blood flow by the kidneys. Incorporation of an iminodiacetic acid (IDA) moiety into PAH yielded p-[(biscarboxymethylaminomethyl)carbamino]hippuric acid (PAHIDA) with a clearance of less than 50% of OIH (Chervu et al., xe2x80x9cTechnetium-99m labeled p-aminohippuric acid analog: A new renal agentxe2x80x9d J. Nucl. Med. 25:1111-1115 (1984)).
Later, the triamide mercaptide (N3S) class of Tc-99m-chelating agents was developed (Fritzberg et al., xe2x80x9cSynthesis and biological evaluation of Tc-99m-MAG3 as a hippuran replacementxe2x80x9d J. Nucl. Med. 27:111-116 (1986)). To date, the 99mTc-mercaptoacetyltriglycine ([99mTcO-MAG3]xe2x88x92) is considered to be one of the most successful agents for functional renal imaging. A few minutes post injection, approximately 1-2% of the injected dose of Tc-99m-MAG3 is found in the kidneys. At the same time, this drug is cleared from the kidney tissue very rapidly. It is the passage into and through the kidneys that provides a measure of renal function (ERPF). Although considered to be the renal imaging agent of choice, Tc-99m-MAG3 is not free of certain problems associated with its use. For instance, the plasma-protein binding of Tc-99m-MAG3 is very high (Taylor et al., Radiology. 162:365-370 (1987); and Bubeck et al., J Nucl. Med. 31:1285-1293 (1990)); the clearance of Tc-99m-MAG3 is only 50-60% of that of OIH and therefore is not suitable for direct measurement of ERPF. In addition, the preparation of Tc-99m-MAG3 requires the kit to be heated at 100xc2x0 C. for 10 min, thus adding an inconvenient step in the preparation.
It was found (Verbruggen et al., xe2x80x9cTechnetium and rhenium in chemistry and nuclear medicinexe2x80x9d, vol.3 (M. Nicolini, G. Bandoli, U. Mazzi, eds.). Verona: Cortina International, pp. 445-452 (1990)) that the polar metabolite of the brain radiopharmaceutical, diethyl Tc-99m-ethylenedicysteine (Tc-99m-L,L-EC), was rapidly and efficiently excreted by the kidneys in mice. This observation prompted the evaluation of Tc-99m-L,L-EC as a potential renal imaging agent. Studies in mice and baboons showed that the pharmacokinetic properties of Tc-99m-L,L-EC are superior to those of Tc-99m-MAG3. Tc-99m-L,L-EC yields a better approximation of ERPF.
The true test of a new radiopharmaceutical, however, is how it performs in patients with various renal disorders that can cause drastic changes in pharmacokinetics. To date a number of clinical studies have been conducted in patients with a variety of renal disorders comparing Tc-99m-L,L-EC and Tc-99m-MAG3. Generally speaking, between the two tracers, there was no significant difference in the image quality or in the parameters derived from the renogram.
Because of the low chemical stability of the thiol group to oxidation, MAG3 is synthesized and supplied in commercial kits as an S-benzyl protected derivative. After reconstitution the kit must be kept in the dark to prevent oxidation of the thiol.
To circumvent this problem, an attempt was made to substitute a hydroxy group for the thiol in MAG3 (Vanbilloen et al., xe2x80x9cCharacteristics and biological behavior of Tc-99m-labeled hydroxyacetylglycine, a potential alternative to 99mTc-MAG3xe2x80x9d.; Eur. J. Nucl. Med. 24:1374-1379 (1997)). The resulting Tc-99m-labeled hydroxyacetyltriglycine (HAG3) had a slightly higher urinary excretion and faster renal transit than Tc-99m-MAG3. The faster renal clearance of Tc-99m-HAG3 can be attributed to its lower plasma protein bindingxe2x80x94comparable with what was seen with Tc-99m-L,L-EC. Although the renal excretion characteristics of Tc-99m-HAG3 are slightly better than those of Tc-99m-MAG3 and the labeling is done at room temperature, the chemical stability of the Tc-99m-HAG3 to transchelation is less than that of the thiol containing analog.
Certain Tc-99m-labeled molecules have a high extraction rate from the bloodstream by the kidneys in combination with a high retention rate by the kidneys. A radiopharmaceutical of this kind is Tc-99m-dimercaptosuccinic acid (Tc-99m-DMSA; Technetium-99m Succimer Injection). The Tc(III) or Tc(V) complex (of unknown structure) is prepared from the reaction of 99mTcO431  with DMSA in the presence of the reducing agent Sn(II) chloride. Tc-99m-DMSA has a specific affinity for the renal cortex. In healthy subjects, the renal activity increases until 6 to 8 hours after injection at which point, a steady level of uptake is reached representing some 30% of the activity administered. At the plasma level, Tc-99m-DMSA is almost completely bound to the proteins. Tc-99m-DMSA is used in morphological studies of kidneys. Such examinations constitute a useful approach in the diagnosis, location and evaluation of various renal pathologies: inflammation, infection, lithiases, traumatisms, tumors etc. Measurement of the renal fixation of the Tc-99m-DMSA complex also makes it possible to assess overall renal function.
A bidentate chelator N-mercaptoacetylglycine (GAM) has been suggested as a Tc-99m-ligand (Gianolli et al., xe2x80x9c99mTc-2GAM: a tracer for renal imagingxe2x80x9d. Nucl. Med. Biol. 23:927-933 (1996)). GAM contains both a thiolato sulfur and an amido nitrogen similar to MAG3 and DADS, but it is a bidentate ligand similar to DMSA. The resulting 2:1 complex (Tc-99m-2GAM) can adopt either a cis- or trans-configuration relative to the oxotechnetium core. Biodistribution studies in animals and normal volunteers indicate that Tc-99m-2GAM has biological properties which are more similar to Tc-99m-DMSA, rather than to Tc-99m-MAG3 or Tc-99m-DADS. Tc-99m-2GAM activity in the kidney reaches a plateau more rapidly than Tc-99m-DMSA and thus may be a possible replacement for Tc-99m-DMSA.
All the above mentioned imaging agents are organic anions. They are transported by the organic anion receptors in the renal tubular system. A buildup of organic anions in plasma, which happens in patients suffering from uremia, can competitively inhibit renal tubular transport of anionic tracers, thereby leading to artificially low estimates of renographic parameters in uremic patients.
Another tubular transport mechanismxe2x80x94the cationic transporter systemxe2x80x94cannot be disrupted by anion accumulation. Tetraazapolyamine chelators, such as cyclam and tetramethylcyclam, were evaluated for their ability to form stable complexes with dioxotechnetium (Herzog et al., xe2x80x9cSynthesis and renal excretion of technetium-99m-labeled organic cationsxe2x80x9d J. Nucl. Med. 33:2190-2195 (1992)). The overall charge of each Tc-99m complex was +1. The magnitude of the plasma protein binding for these organic cations was comparable to that of Tc-99m-EC and significantly less than that of OIH and Tc-99m-MAG3. The renal clearance of tetraazapolyamines was similar to that of Tc-99m-L,L-EC. The mode of excretion of these tracers by the tubule cationic transporter system was clearly identified.
The choice of Tc-99m-radiopharmaceuticals for renal imaging is presently limited to a few small hydrophilic molecules containing mostly N2S2 technetium cores. Within each class of existing kidney radiotracers there are not many possibilities for modifying the periphery of the molecule to fine tune the solubility of the compound and its pharmacokinetics. Therefore, there is a need to develop new Tc-99m ligands which could vary the size and lipophilicity of stable Tc-99m-complexes containing the same type of Tc-core.
The present invention provides metal chelates which are suitable as pharmacutical imaging agents for various organs and tissues, preferably imaging renal tissues. The metal chelates of this invention have high organ-specificity for kidney tissues. The chelates possess sufficient stability to allow the completion of the preparation of the radiopharmaceutical, as well as a thorough performance of a renal examination.
In particular embodiments of this inventions, the metal chelates based on a 2-pyrrolylthione structure are provided as being suitable as radiopharmaceutical agents and represented by Formula I: 
wherein
M is independently selected from the group consisting of radioisotopes of Tc, Re, Cd, Pb, Zn, Ag, Au, Ga, Pt, Pd, Rh, Cr, Cu, V and the like;
n=1 to 4;
R1, R2, and R3 is independently hydrogen, alkyl, OH or its derivative, halogen, NO2, NH2, N+R3, NHCOR, CN, an alkyl carboxylic acid or acid ester group or its derivative, keto, SO3H or its derivative, or a group that, when taken together with another ring, ring substituent, forms a fused 5 or 6 membered ring, wherein R is independently hydrogen, alkyl, OH or its derivative, halogen, CN, an alkyl carboxylic acid or acid ester group or its derivative, keto, or SO3H or its derivative;
unsubstituted or substituted alkyl or heteroalkyl, unsubstituted or substituted carbocycle, including aryl, unsubstituted or substituted heterocycle, AOH, ACOOH, ACOOR, AHal, CN, ANO2, ANH2, ANR2, AN+R3, and ANHCOR wherein A is alkyl, heteroalkyl, carbocycle, including aryl or heterocycle, and R is alkyl or aryl and Hal is a halogen, preferably F, CL, Br, or I.
Thus the metal chelates also include from one to four 2-pyrrolylthiones bound to a single metal atom or isotope. Moreover, the chelates contain variations in the ligand periphery, encompassed by positions R1, R2, R3, and X above, to contain hydrophilic or lipophilic substituents.
In one aspect of the invention, the di-2-pyrrolylthione-based metal chelates encompassed by Formula I have the desirable characteristics of significant renal uptake and retention. Preferably, such characteristics are comparable, or superior to, that observed with Tc-99m-DMSA (Technetium-99m Succimer Injection). Moreover, the metal chelates of the invention permit the resulting diagnostic image quality to be comparable or superior to that with Tc-99m-DMSA (Technetium-99m Succimer Injection).
In another embodiment of the invention, Tc-99m-chelates encompassed by Formula I are available in high radiochemical purity ( greater than 90%) and with high specific activity.
In another aspect of the invention, methods used to synthesize Tc-99m chelates encompassed by Formula I are disclosed. The methods used to purify such radiopharmaceuticals are also described. Furthermore, this invention provides a radiopharmaceutical composition useful for renal imaging and as a therapeutic. Such a radiopharmaceutical composition is comprised of a metal chelate as described above and a pharmaceutically acceptable carrier.
Particular embodiments of this invention provide 2-pyrrolylthione ligands suitable for the formation of metal chelates of Formula I. These ligands are encompassed by the following Formula II: 
wherein
R1, R2, and R3 is independently hydrogen, alkyl, OH or its derivative, halogen, NO2, NH2, N+R3, NHCOR, CN, an alkyl carboxylic acid or acid ester group or its derivative, keto, SO3H or its derivative, or a group that, when taken together with another ring, ring substituent, forms a fused 5 or 6 membered ring, wherein R is independently hydrogen, alkyl, OH or its derivative, halogen, CN, an alkyl carboxylic acid or acid ester group or its derivative, keto, or SO3H or its derivative;
X is independently selected from the group consisting of unsubstituted or substituted alkyl or heteroalkyl, unsubstituted or substituted carbocycle, including aryl, unsubstituted or substituted heterocycle, AOH, ACOOH, ACOOR, AHal, CN, ANO2, ANH2, ANR2, AN+R3, and ANHCOR wherein A is alkyl, heteroalkyl, carbocycle, including aryl or heterocycle, and R is alkyl or aryl and Hal is a halogen, preferably F, CL, Br, or I.
Particular embodiments of this invention include 2-pyrrolylthione ligands encompassed by Formula II that are capable of forming metal chelates with nuclides including, but not limited to, technetium, rhenium, cadmium, zinc, lead, silver, gold, gallium, platinum, palladium, rhodium, chromium, vanadium and the like.
The present invention also includes specific procedures for the chelation process between a nuclide and a ligand satisfying Formula II, thereby forming a metal chelate that has a chemical structure specified by Formula I.
It is further contemplated in this invention that the ligands of Formula II can be utilized for inhibiting metalloenzymes and for metal chelation, particularly chelation of toxic metals.
The present invention also provides kits that incorporate the features of the invention and makes possible a convenient means of practicing the invention. Kits of the invention comprise one or more compounds and/or compositions as described herein and may also include other materials that facilitate the practice of the invention, such as, but not limited to, instructions, descriptive indicators or labels, and devices relating to administration and/or use of the kit contents. The items comprising the kit may be supplied in the form of individual packages and/or packaged together, as desired by the skilled practitioner.